Publication details
Zmapovanie interakcií kolicínov U a Y s receptorom OmpA
| Title in English | Mapping the interactions of colicins U and Y with their receptor OmpA |
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| Authors | |
| Year of publication | 2008 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | OmpA (outer membrane protein A) is an important protein of gramnegative bacteria cell envelope. It contains two domains transmembrane b-barrel and periplasmic globular C domain. It is supposed the importance of this protein in connection of outer membrane with rigid peptidoglycan layer. OmpA acts also as a receptor for many antibacterial agens bacteriophages and colicins. OmpA interacts with four colicins U, Y, K, and L. For colicins U and Y, OmpA has a major receptor function, whereas for colicin K and L, it is only a protein helping with transport. Bacteria of strains Escherichia coli and Enterobacter aerogenes differ in their sensitivity to colicins U and Y. These two strains have variability in OmpA sequence. Variable amino acids of OmpA extracellular loops were distributed into 11 segments. Every segment was built of 1 to 3 amino acids. We used PCR in mutagenesis of OmpAs segments. We proposed 11 pairs of primers. Every primers pair has a specific sequence from insensitive OmpA of E. aerogenes. By PCR mutagenesis, it was amplified a plasmid with ompA E. coli (only sequence of b-barrel), which was originally sensitive to colicins. Constructs were verifying by sequencing and the correct plasmids with targeted mutation were transformed in E. coli BL21 (lDE3ompA-). Then we provide a sensitivity of strains with mutant OmpA to colicins U and Y. The decrease of sensitivity to colicin U was observed in strains E. coli with mutation in OmpA segments 1, 2, 3, 7 and 9. By colicin Y, sensitivity was reduced in strains with mutation in segment 1, 2, 7, 9. The strong decrease of sensitivity was upon mutagenesis of segments 1 or 2 and weaker decrease of sensitivity was observed after mutation in segments 3, 7 or 9. This shows a different intensity of interaction. For identified segments 1, 2, 3, 7 and 9, PCR mutagenesis was done by analogous way, where simple amino acids were replaced. For segments 1.1 and 2.2, we observed a decrease of sensitivity. These two segments present amino acids isoleucin I45 and glycin G48. Change of one amino acid in segments 3, 7 or 9 has no effect on sensitivity to colicin. We suppose, the reason is a very small decrease of sensitivity or ability of other interacting amino acids to compensate the change of one amino acid. Colicins interact with receptor OmpA on various extracellular loops, and these interactions join several amino acids with various intensity. |
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